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Bacteriology

Single-Molecule Long-Read 16S Sequencing To Characterize the Lung Microbiome from Mechanically Ventilated Patients with Suspected Pneumonia

Ian Toma, Marc O. Siegel, John Keiser, Anna Yakovleva, Alvin Kim, Lionel Davenport, Joseph Devaney, Eric P. Hoffman, Rami Alsubail, Keith A. Crandall, Eduardo Castro-Nallar, Marcos Pérez-Losada, Sarah K. Hilton, Lakhmir S. Chawla, Timothy A. McCaffrey, Gary L. Simon
G. V. Doern, Editor
Ian Toma
aDepartment of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
hDepartment of Physical Therapy and Health Care Sciences, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Marc O. Siegel
bDivision of Infectious Diseases, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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John Keiser
dDepartment of Pathology, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Anna Yakovleva
aDepartment of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Alvin Kim
aDepartment of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Lionel Davenport
eChildren's National Medical Research Center, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Joseph Devaney
eChildren's National Medical Research Center, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Eric P. Hoffman
eChildren's National Medical Research Center, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Rami Alsubail
aDepartment of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Keith A. Crandall
fComputational Biology Institute, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Eduardo Castro-Nallar
fComputational Biology Institute, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Marcos Pérez-Losada
fComputational Biology Institute, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
iCentro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), Vairão, Portugal
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Sarah K. Hilton
fComputational Biology Institute, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Lakhmir S. Chawla
gDepartment of Anesthesiology, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Timothy A. McCaffrey
aDepartment of Medicine, Division of Genomic Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
cDepartment of Microbiology, Immunology, and Tropical Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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Gary L. Simon
bDivision of Infectious Diseases, The George Washington University School of Medicine and Health Sciences, Washington, DC, USA
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G. V. Doern
Roles: Editor
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DOI: 10.1128/JCM.01678-14
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ABSTRACT

In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles.

FOOTNOTES

    • Received 20 June 2014.
    • Returned for modification 16 July 2014.
    • Accepted 18 August 2014.
    • Accepted manuscript posted online 20 August 2014.
  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.01678-14.

  • Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Single-Molecule Long-Read 16S Sequencing To Characterize the Lung Microbiome from Mechanically Ventilated Patients with Suspected Pneumonia
Ian Toma, Marc O. Siegel, John Keiser, Anna Yakovleva, Alvin Kim, Lionel Davenport, Joseph Devaney, Eric P. Hoffman, Rami Alsubail, Keith A. Crandall, Eduardo Castro-Nallar, Marcos Pérez-Losada, Sarah K. Hilton, Lakhmir S. Chawla, Timothy A. McCaffrey, Gary L. Simon
Journal of Clinical Microbiology Oct 2014, 52 (11) 3913-3921; DOI: 10.1128/JCM.01678-14

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Single-Molecule Long-Read 16S Sequencing To Characterize the Lung Microbiome from Mechanically Ventilated Patients with Suspected Pneumonia
Ian Toma, Marc O. Siegel, John Keiser, Anna Yakovleva, Alvin Kim, Lionel Davenport, Joseph Devaney, Eric P. Hoffman, Rami Alsubail, Keith A. Crandall, Eduardo Castro-Nallar, Marcos Pérez-Losada, Sarah K. Hilton, Lakhmir S. Chawla, Timothy A. McCaffrey, Gary L. Simon
Journal of Clinical Microbiology Oct 2014, 52 (11) 3913-3921; DOI: 10.1128/JCM.01678-14
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